MICROBIOLOGY

ENUMERATION OF BACTERIA
Instructor St.Peters Institute of Pharmaceutical Sciences Vidyanagar, Hanamkonda, India

Introduction
To be specific, the count actually represents the

number of Colony Forming Units (cfu) per g ( or per ml) of the sample.

Classification of counting techniques

Counting

Direct

Indirect

Viable count

Total count
Turbidity Metabolic activity

Dry mass

Most probable number

Plate counts

Spread plate method

Pour plate method

Methods of counting bacteria

Direct count Actual counting of microorganisms:
A method by which a reasonably statistically precise

measurement of cell quantification is achieved. The basis of a direct count is the actual counting of every organism (or every living organism---often limited to a single type of microorganism ) present in a sub-sample of a population.

Direct counts include:

Plate counts (a viable count) Most probable number method (a viable count) Direct microscopic count (a total count)
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DIRECT COUNT
Dilutions of samples are observed under a

microscope the number of bacterial cells from a given volume of sample are counted

DIRECT COUNT

DIRECT COUNT

Cell density is determined by:

1) Counting the number of bacteria in one small square

2) Multiplying by the dilution factor of the sample

Dilution factor of 107 Number of bacteria in square = 5 5 X 107 = 50,000,000 bacteria per ml

Merits
1)The technique is sensitive and has the advantage of only

counting living bacteria, 2) Any concentration of microorganism can be easily counted, if the appropriate dilution is plated 3) It is even possible to concentrate a solution before counting, as is often done in water analysis, where bacterial populations are usually at low density. 4) The equipment necessary for performing viable plate counts is readily available in any microbiology lab and is cheap in comparison to other methods. 5) Finally, by using a selective medium it is possible to determine the number of bacteria of a certain class, even in mixed populations.

Demerits
One major disadvantage of the viable plate count is the

assumption that each colony arises from one cell. In species where cells grow together in clusters, a gross underestimation of the true population results. One example of this are species of Staphylococcus, which is known to form clumps of microorganisms in solution. 2)t he temperature of incubation and medium conditions must also be optimized to achieve the largest colonies possible so that they are easily counted. Finally, this technique takes time. Depending on the organism, one day to several weeks might be necessary to determine the number of CFUs that were present when the experiment started. Such information may no longer be useful for many experiments.
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Methods of counting bacteria contd.

Viable count A viable count is a direct counting method in which only
Pour plating Spread plating Most probable number method

viable cells are counted. Viable counts can be accomplished by such techniques as:

Total count A total count is a direct counting method in which all cells

are counted, whether dead or alive . Generally taking total counts requires the employment of microscopes.

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Note
A sample to be counted is diluted in a solution that will not harm the microbe, yet does not support its growth (so they do not grow during the analysis).

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Spread-plate technique
In the spread-plate technique some of the highest dilutions (lowest bacterial density) are then taken and spread with a sterile glass rod onto a solid medium that will support the growth of the microbe. It is important that the liquid spread onto the plate soaks into the agar. This prevents left over liquid on the surface from causing colonies to run together and the need for dry plates restricts the volume to 0.1 ml or less.

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pour plate technique

A second method for counting viable bacteria is the pour plate technique, which consists of mixing a portion of the dilution with molten agar and pouring the mixture into a petri plate. In either case, sample dilution is high enough that individual cells are deposited on the agar and these give rise to colonies. By counting each colony, the total number of colony forming units (CFUs) on the plate is determined. By multiplying this count by the total dilution of the solution, it is possible to find the total number of CFUs in the original sample
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VIABLE PLATE COUNT

TNTC
if the number of colonies is too great (over 300) the

sample is labeled TNTC Too Numerous To Count

limitation of viable plate count selective as to the bacterial types that will grow given the incubation temperature and nutrient type

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 Plate counts [pour plate, spread plate] Colonies founded by cells:

Colonies grown in petri dishes by various methods (not including streaking )

may be used to determine the count of viable microorganisms . Plate counts assume that every colony is founded by a single cell. That cell must have been alive in order to grow and form a colony .

Caveats:
Problems with plate counts are:

they require lengthy incubation for colonies to become visible cell clumping can lead to an undercount of viable cells it is very easy to have too many or too few colonies on a plate to accurately measure viable count prevention of crowding often requires serial dilution* too few cells requires concentrating, e.g., by centrifugation or filtration** *Depending on your organism, and other circumstances, you will want to have no more than 300 to 500 colonies per plate (e.g., for big colonies 300 will be many, for small colonies 500 may still work).

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MOST PROBABLE NUMBER

Statistical method based on probability theory

multiple serial dilutions are performed to reach a point

of extinction
dilution level at which no cells are deposited

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MOST PROBABLE NUMBER

Criteria have been developed for indicating whether a

dilution tube has bacteria present the pattern of negative results compared statistically results

positive and are with a table of probabilities for

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MOST PROBABLE NUMBER

The pattern to tubes

that show growth (brown) and those that do not (orange) are compared with a statistical table to calculate MPN

MOST PROBABLE NUMBER

MPN of the original sample is 170 per 100 mL

MOST PROBABLE NUMBER

Non plate viable count:
The most probable number method is a way of determining approximate viable count by

diluting cultures then growing the dilution cultures in broth tubes. At the dilution at which broth becomes turbid or not with similar likelihood, the culture has been dilution to the point that the broth tubes were inoculated with on the order of only a single microorganism (turbid ) or fewer (not turbid ). The concentration of the culture is then taken to be equal to the amount of dilution necessary to have reached this point.

Advantages
MPN is especially useful in situations where there is an advantage to using broth over solid

medium . For example, many organisms are not good at forming colonies, such as highly motile organisms.

"When samples contain too few organisms to give reliable measures of population size by the standard plate count method, as in food and water

sanitation studies, or when organisms will not grow on agar, the most probable number method is used."

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Total count
Direct microscopic count Direct microscopic count is a determination of the number

of microorganisms found within a demarcated region of a slide known to hold a certain volume of culture . This total count method of cell quantification is very rapid but has the problem of requiring high cell concentrations (e.g., 107 / ml) as well as potentially counting dead and living cells with equal probability.

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Indirect methods
Estimation of cell number Correlates to cell number:
Estimates of cell number use various correlates of cell number

rather than direct counting . Such methods are often preferable either for convenience or because direct counting is difficult or even impossible in many situations (for example, when quantifying filamentous organisms ).

Estimates of cell number include determinations of:

Turbidity Metabolic activity Dry mass
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Methods of counting bacteria contd.

Filtration (Concentrating cells):
If too few colonies are present then the original culture

must be concentrated prior to determining its plate count. Filtration, which sieves microorganisms out of the medium, is one method of concentrating microorganisms .

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Methods of counting bacteria contd.

Turbidity The cloudiness or turbidity of a culture is caused by the individual cells scattering light. Degree of turbidity is a direct correlate of cell mass. Estimator of cell number:
The average cell mass of individual cells in a culture must first be determined if the

turbidity of a culture is to be used to estimate cell count . This standardization usually works best for cultures growing in exponential phase .

Metabolic activity The metabolic output or input of a culture may be used to estimate viable count. This method has the same caveats as turbidity methods. Examples include: "The rate at which metabolic products such as gases and/or acids are formed by culture reflects the mass of bacteria present. . . The rate at

which a substrate such as glucose or oxygen is used up also refelcts cell mass.

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Methods of counting bacteria contd.

Dry mass Estimation of cell count:
Determinations of dry mass has all of the problems of turbidity and metabolic

activity with the added problem of having to dry cultures before employing it. "To calculate the dry weight of cells, they must be separated from the medium by some physical means such as filtration or centrifugation. The cells are then dried, and the resulting mass is weighed

For filamentous organisms this method works sufficiently well

compared to the other methods listed that dry mass determination is employed.
When clumped both or more cells are counted as only a single cell in a viable

Counted as single cell:

count. This is one disadvantage of employed viable counts for cell quantification .

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